ID #268 Brain-sensing synNotch-CAR T-cells enable precise targeting and clearance of diffuse midline glioma in multiple preclinical models
Senthilnath Lakshmanachetty, Milos Simic, Payal Watchmaker, Jeffrey Haegelin, James Long, Aishi Zhao, Robert Zhu, Maggie Colton Cove, Alejandra Bergquist, Brianna Wong, Marco Gallus, Takahide Nejo, Ricardo Almeida, Joanna Phillips, Wendell Lim, Hideho OkadaAbstract
Background
Diffuse midline glioma (DMG), including diffuse intrinsic pontine glioma (DIPG), is a fatal pediatric brain tumor with limited therapeutic options. Although early-phase clinical trials indicate promise for chimeric antigen receptor (CAR) T-cell therapy, the lack of tumor-specific antigens, poor trafficking, and limited T-cell persistence remain significant barriers to achieving meaningful clinical benefit.
Methods
We engineered a synthetic Notch (synNotch)–based “prime-and-kill” CAR T-cell circuit in which recognition of the brain-specific proteoglycan, Brevican (BCAN) induces expression of a tandem CAR targeting B7-H3 and IL13Rα2, antigens highly expressed in DMG. This brain-sensing strategy restricts CAR expression to the tumor microenvironment while minimizing peripheral off-tumor activity.
Results
α-BCAN synNotch→α-B7-H3/IL13Rα2 (B-SYNC-B/I) T-cells demonstrated potent, BCAN-dependent cytotoxicity against DMG/DIPG cell lines in vitro. A single intravenous infusion significantly prolonged survival in immunodeficient mice bearing orthotopic DMG xenografts, with complete tumor eradication observed in approximately 50% of treated animals. In contrast, intracerebroventricular (ICV) delivery of B-SYNC-B/I T-cells caused significant neurotoxicity in both tumor and non-tumor-bearing mice, even at low doses, consistent with on-target, off-tumor recognition of B7-H3 expressed on normal brain tissue. In immunocompetent DMG models, ICV infusion of B-SYNC-B7-H3 T-cells with or without lymphodepletion led to durable tumor eradication without neurotoxicity and conferred protection against tumor rechallenge. Conversely, constitutively active B7-H3/IL13Rα2 CAR T cells failed to persist, traffic to the brainstem, or improve survival in immunodeficient models; however, lymphodepletion allowed effective tumor clearance following ICV delivery of constitutive B7-H3 CAR T-cells in immunocompetent mice.
Conclusions
Brain-sensing synNotch-CAR T-cells provide spatially restricted activation and robust anti-tumor efficacy in preclinical DMG models, supporting their clinical evaluation as a promising therapeutic strategy for pediatric patients with DMG/DIPG.